Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production

The number of available promoters for the protein production host Pichia pastoris is limited, and in most applications comprises the methanol inducible alcohol oxidase 1 (AOX1) promoter and the constitutive glyceraldehyde phosphate dehydrogenase (GAP) promoter. To close this gap, we identified 24 novel potential regulatory sequences and tested their applicability to drive the expression of both intracellular as well as secretory heterologous proteins. While more than 80% of the promoters derived from microarray data mining showed activity on all common carbon sources used for P. pastoris, the success rate of rationally selected promoters was lower. Many of the newly identified promoters showed a growth rate dependent behaviour, for example three ribosomal promoters as well as the promoters of two chaperones were much more active at early growth phase as compared to later phases. Fed batch cultivation of selected promoters expressing human serum albumin further strengthened the correlation of promoter activity (determined by HSA transcript levels) to specific growth rate. The promoter of the thiamine biosynthesis gene PTHI11 did not only show high transcript levels at low specific growth rate, but also exhibited interesting regulatory properties dependent on the availability of thiamine in the growth medium.